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2017-Rahman et al-Cytometry B Clin Cytom

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mleipold

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Post Mon Jul 31, 2017 3:15 pm

2017-Rahman et al-Cytometry B Clin Cytom

"High-Dimensional Single Cell Mapping of Cerium Distribution in the Lung Immune Microenvironment of an Active Smoker"
Rahman AH, Lavin Y, Kobayashi S, Leader A, Merad M
Cytometry B Clin Cytom. 2017
http://dx.doi.org/10.1002/cyto.b.21545
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mleipold

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Post Mon Jul 31, 2017 5:38 pm

Re: 2017-Rahman et al-Cytometry B Clin Cytom

Hi Adeeb,

I have a technical question on the data analysis. The Methods mention the use of the EQ normalization beads, as usual, and that you normalized using the Fluidigm normalizer.

Did you notice whether you had any issues with EQ beads remaining in your data files after normalization, since you had the Ce140+ cells? Or, conversely, "losing" some of your Ce140+ cells of interest from the normalization?

I know that you *shouldn't*, since the cells would be dim for the other 3 elements (and, at ~250 Dual in Fig 1A and 1E, dimmer than the usually >1000 Dual Ce140 in the beads). But since this is the first time I'm aware of someone having a strong Ce140 signal, I thought I would ask.
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AdeebR

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Post Mon Jul 31, 2017 8:11 pm

Re: 2017-Rahman et al-Cytometry B Clin Cytom

Hi Mike,

I actually never remove the beads when using the normalizer - I prefer to leave them in the file and gate them out during analysis. I will say that when we first came across this phenomenon, we missed it because the Ce140 high cells were excluded when performing the standard stringent Ce140 vs DNA gate to exclude residual beads/bead-cell doublets. I realized later that we had a couple of samples with surprisingly few macrophages and when I went back and reviewed the data I noticed the Ce140 positivity in the macrophage population. So for this experiment, I ended up excluding the beads on the basis of Eu151/153 instead of Ce140.

Adeeb
Adeeb Rahman
Icahn School of Medicine at Mount Sinai, NYC
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AdeebR

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Post Mon Jul 31, 2017 8:24 pm

Re: 2017-Rahman et al-Cytometry B Clin Cytom

Incidentally, I don't know if you noticed the formal shout out in the acknowledgements:

"...We also thank Mike Leipold and Christoph Schwärzler for their insightful discussions and contributions on CyTOF Forum on a wide range of topics including mass cytometry background contamination".


I credit my use of the term "Mischmetal" in this manuscript largely to this post by Cristoph:

ChristophS wrote:Hi Astrid,

since no single element would have an isotope composition that could explain this pattern it looks a bit like the BSA you use may have traces of Mischmetal (https://en.wikipedia.org/wiki/Mischmetal) which typically is composed of a majority of Cerium (you should see ~8% of your 140 signal in 142) and other lanthanides. It is used in lighter flints and (wild hand-waving) the cattle may have ingested one of those or the pastures are in the vicinity of mines or production plants.

Hope this may help to shed light on the problem. BTW did you check alternative sources of BSA?

Christoph


From this thread: viewtopic.php?f=4&t=688&hilit=cerium
Adeeb Rahman
Icahn School of Medicine at Mount Sinai, NYC
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AdeebR

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Post Wed Aug 02, 2017 1:55 am

Re: 2017-Rahman et al-Cytometry B Clin Cytom

FCS files (including tSNE parameters) for total CD45+ cells and gated subpopulations from blood, lung and tumor are available here:

https://flowrepository.org/id/FR-FCM-ZY9B
Adeeb Rahman
Icahn School of Medicine at Mount Sinai, NYC
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mleipold

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Post Thu Aug 01, 2019 6:36 pm

Re: 2017-Rahman et al-Cytometry B Clin Cytom

Related paper since publication of 2017-Rahman et al:

"Relationship between domestic smoking and metals and rare earth elements concentration in indoor PM2.5"
https://doi.org/10.1016/j.envres.2018.03.026

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